PROJECT SUMMARY The goal of this proposal is to develop assays for cardiotoxicity and hepatotoxicity using magnetic 3D bioprinting. Both cardiotoxicity and hepatotoxicity are critical areas for safety testing, and in need of more accurate in vitro screens to prevent costly market withdrawals. However, current in vitro options are lacking in throughput and representation of native tissue environments. This proposal uses magnetic 3D bioprinting to create 3D in vitro culture models that mimic native tissue. The principle behind magnetic 3D bioprinting is the magnetization of cells and their printing using magnetic forces. This method is rapid and easy to use, and escapes the technical issue of other 3D culture models that limit its use for high-throughput screening. In Phase I, a general toxicity assay, the BiO Assay, was developed using magnetically 3D bioprinted spheroids. This Phase II builds on that success, by developing this cytotoxic assay for cardiomyocytes and hepatocytes, then further developing tissue-specific functional assays to determine the effect of compounds on tissue function. For cardiomyocytes, a beating assay will be developed to assay a compound's effect on beating, while for hepatocytes, an assay for cytochrome P450 induction/inhibition will be developed to assay a compound's effect on metabolism. In combination with other assays, such as LC-MS and fluorescent calcium signaling assays, our assays will offer high-content and high-throughput solutions to study both cytotoxicity and functional toxicity. This work aids NIEHS in its goal of finding in vitro solutions for toxicity screening that are high-throughput and representative of native tissue environments. The end result of this proposed work is a full suite of assays to study a compound's cardiotoxic and hepatotoxic effects.